CN112680442B - Peanut wild species genome specific probe and use method - Google Patents
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Abstract
The invention relates to a peanut wild species genome specific probe and a use method thereof, wherein the peanut wild species is subjected to illuminea double-end genome sequencing to obtain a genome sequence with the depth of 20 multiplied by the depth, and the peanut wild species specific repetitive sequence probe is designed by bioinformatics software analysis to obtain H genome specific probes Dard-127 and Pus-187 and B genome specific probes Ipa-163. The genome specific probe is utilized to prepare probe dye liquor to dye peanut chromosomes, and the probes Pus-187 are H genome specific probes, so that H genome species can be specifically identified; probe Dard-127 is an a.dardonoi specific probe capable of specifically recognizing a.dardonoi; probes Ipa-163 are B and K genome specific probes, suggesting that B and K genomes may be closely related. The invention provides a cytological marker and a method capable of effectively identifying peanut genome or specific wild species, and provides a new method and theoretical basis for revealing the genetic relationship between peanut wild species or genomes, researching peanut interspecific hybridization and utilizing wild species.
Description
Technical Field
The invention relates to a peanut cytogenetic research method, in particular to a design and use method of a peanut wild species genome specific probe.
Background
The wild peanut has rich biological and abiotic stress resistance sources, high oil content and other high quality characters, and the resistance mechanism is different from that of the peanut cultivated species, so that the wild peanut is an important gene resource for improving the peanut cultivated species. Research at home and abroad shows that more than 120 plant diseases and insect pests damaging peanuts cause 5% -100% of yield loss; peanut wild species have a high level of resistance to many peanut diseases, insect pests and growth stress, almost all peanut diseases can find sources of resistance in peanut wild species, some wild species are even immune to some diseases, such as a.digoi and a.glabra against peanut bud necrosis virus disease and tomato spotted wilt virus, a.kuhlmannii, a.duranensis and a.ipaensis against peanut cluster virus disease, a.magna and a.cadensii against rust disease, etc., which cannot be found in peanut cultivars.
Interspecific crossing is an important means for introducing excellent genes of peanut wild species into cultivars. However, peanut species have been shown to have different degrees of reproductive isolation due to differences in chromosomal ploidy, genomic composition, etc., by hybridization of species within the same genome or between different genomes, for example: cross incompatibility, hybrid death, hybrid sterility and other cross disorders, which result in different difficulties in introducing genes of different genomic wild species into cultivars. It was found that the species of peanut cultivars (genomic AABB) hybridized with the A, B, K, F, G isogenomic species in the peanut group (section Arachis) were almost compatible, and that hybridization with the E genome-possessing partial upright group (section Erectoides) species and the R genome-possessing partial rhizome group (section Rhizomatosae) species was also easier; whereas species crossing with the large root group with the C genome (section Caulorrhizae), the creeping group with the P genome (section Procumbentes), the trefoil group with the Te genome (section Trierectoides), the trinung group with the T genome (section Triseminatae), the surrounding vein group with the Ex genome (section Extranervosae) and the odd-shaped flower group with the H genome (section Heteranthae) were difficult to succeed. Therefore, the definition of the correlation between peanut wild species genomes is important to overcome the incompatibility obstacle of peanut interspecific hybridization and the efficient utilization of excellent wild species.
In the evolution process of plant genome, the repeated sequences have different degrees of amplification and contraction, and the amplification and the contraction cause the types and the contents of the repeated sequences to be different between species and inside the species, and the difference can reflect the relatedness between the species. Tandem repeat sequences are repeated sequences distributed in clusters in a genome, and amplification of the tandem repeat sequences provides a rich source of variation for genome evolution. Plant-specific tandem repeats are usually amplified only in closely related species or genomes, but not in other genomes, and are therefore often used to determine relatedness between species or genomes.
Peanut wild species undergo a lengthy evolution process, forming different genome types such as A, B, K, F, G, C, P, T, te, ex and H. However, no study on specific repeated sequences of peanut wild genome has been reported.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a peanut wild species genome specific probe and a using method, wherein H genome species A.dardonoi and A.pusilla and B genome species A.ipaensis are taken as research objects, the peanut wild species genome specific probe is established through genome sequencing and bioinformatics analysis, and is applied to peanut wild species genetic relationship analysis, so that a novel method and theoretical basis are provided for peanut distant hybridization research and wild species utilization.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
a genome specific probe of a peanut wild species, which comprises an H genome specific probe and a B genome specific probe; the H genome specific probes are Dard-127 and Pus-187, the B genome specific probes are Ipa-163, and the specific sequences are as follows:
probe Dard-127:
FAM-5′-TAAACTATGGTATTTTCATGAGTTTTGAGGCATGCCGGA-3′；
probes Pus-187:
TAMRA-5′-TCACTAGGCATATAATGCCACTCGATGGCGTTGAAACGCGGAGCT-3′；
probes Ipa-163:
TAMRA-5′-TAGGGTTTATGATTTAGGCTTTAGGGTTTGT-3′。
the design method of the peanut wild species genome specific probe comprises the following steps:
(a) Firstly carrying out illuminea double-end genome sequencing on peanut species, wherein the sequencing depth is 20×; then, the genome sequencing original sequence is filtered by utilizing Trimmomatic software, and key parameters are as follows: ILLUMINACIP: TRIM_HOME/adapters/TruSeq3-PE.fa:2:30:10, LEADING:5, TRAILING:5 SLIDINGWINDOWN: 4:20MINLEN:150, to obtain high quality "clean reads";
(b) Using a peanut cultivar Tifrunner genome as a reference genome, constructing an index file by using bowtie2 software, then comparing 'clean reads' to the Tifrunner reference genome, and filtering 'clean reads' of which the two ends are not compared to the Tifrunner reference genome;
(c) Randomly sampling 100 ten thousand sequences from filtered clean reads by utilizing a shuf command of Linux, and utilizing TAREAN software to perform tandem repeat sequence discovery on the clean reads obtained based on random sampling to discover a tandem repeat sequence set of high confidence;
(d) Positioning the tandem repeat sequence onto a peanut cultivar tifrenner reference genome using B2DSC, excluding the tandem repeat sequence highly enriched in cultivars; using NCBI-BLAST to exclude possible gene sequences of candidate tandem repeat sequences, and using the remaining tandem repeat sequences for probe design;
(e) The most conserved segment of the tandem repeat sequence is selected to intercept the sequence of 30-45bp, and 5' -TAMRA or FAM is artificially synthesized and modified to obtain the genome specific probe.
The peanut species are H genome species A.dardonoi and A.pusilla, and the corresponding genome specific probes are H genome specific probes Dard-127 and Pus-187.
The peanut species is the B genome species A.ipaensis, and the corresponding genome specific probe is the B genome specific probe Ipa-163.
The application method of the peanut wild species genome specific probe comprises the following steps:
(a) Chromosome tabletting to obtain metaphase chromosome tablets of root tip cells of diploid wild species with peanut genome A, peanut genome B, peanut genome E, peanut genome H and peanut genome K, respectively, freezing at-80 ℃, removing tablets, dehydrating with absolute ethyl alcohol, and air-drying;
(b) 40mL of 2 XSSC buffer solution, H genome specific probe dry powder or/and B genome specific probe dry powder and 5 mu L of DAPI mother solution are added into a dye vat, a wild chromosome slice is placed into the dye vat for dyeing for 5 hours, and a fluorescent microscope is used for photographing; the H genome specific probe dry powder is 0.1OD Dard-127 and 0.1OD Pus-187, and the B genome specific probe dry powder is 0.1OD Ipa-163.
2 XSSC buffer from 0.3mol/L trisodium citrate C 6 H 5 Na 3 O 7 ·2H 2 O and 3mol/L NaCl.
DAPI mother liquor was DAPI staining solution at a concentration of 100. Mu.g/mL.
The wild species of the A genome, the B genome, the E genome, the H genome and the K genome are respectively A genome species A.duraensis, A genome species A.diogoi, B genome species A.ipaensis, E genome species A.paralogiensis, E genome species A.steptophla, H genome species A.pusilla, H genome species A.dardonoi and K genome species A.bat zocoi.
The invention has the beneficial effects that:
according to the invention, through carrying out illumina double-end genome sequencing on peanut wild species, genome sequence and bioinformatics analysis are utilized, specific probe markers of peanut wild species H and B genomes are successfully developed, the cytologic characteristics of the wild species are enriched, a design method of a peanut wild species genome specific probe is established, and a novel effective method is provided for obtaining peanut wild species genome specific cytologic markers.
The invention utilizes newly designed peanut wild species H and B genome specific probes to carry out chromosome staining analysis on a plurality of peanut genomes, and discovers that probes Pus-187 are H genome specific probes and can specifically identify H genome species; probe Dard-127 is an a.dardonoi specific probe that can specifically recognize a.dardonoi; probes Ipa-163 are B and K genome specific probes, confirming that B and K genomes may be closely related. The specific probe can be used for analyzing different peanut wild species genomes, can effectively identify peanut wild species or genomes, determines the genetic relationship between peanut wild species or genomes, and provides a novel method and theoretical basis for peanut interspecific hybridization research and wild species utilization.
Drawings
FIG. 1 shows the result of staining signals with specific probes Pus-187 and Dard-127 on wild species A.duraensis (a, A genome), A.digoi (B, A genome), A.ipaensis (c, B genome), A.paralogiensis (d, E genome), A.stenophylla (E, E genome), A.pusilla (f, H genome), A dardonoi (g, H genome) and A.bat zocoi (H, K genome).
Wherein, the first column from left to right in (a-h) is DAPI staining, the second column is Pus-187 probe red signal distribution on wild type chromosome, the third column is Dard-127 probe green signal on wild type chromosome, and the fourth column is DAPI, pus-187 and Dard-127 probe signal synthesis map.
FIG. 2 shows the result of staining signals with specific probes Ipa-163 on wild species A.duraensis (a, A genome), A.diogoi (B, A genome), A.ipaensis (c, B genome), A.paraguariensis (d, E genome), A.stenophylla (E, E genome), A.dardonoi (f, H genome) and A.bat zocoi (g, K genome).
Wherein, the first column from left to right in (a-h) is DAPI staining, the second column is distribution of the red signal of the Ipa-163 probe on the wild-type chromosome, and the third column is a synthetic diagram of DAPI and the red signal of the Ipa-163 probe.
Detailed Description
The following describes the embodiments of the present invention in further detail with reference to examples.
Example 1, design method of peanut wild species H genome specific Probe
(a) Two H genome species A.dardonoi and A.pusilla were first submitted to illuminea double ended genome sequencing to a depth of 20×. The genome sequencing original sequence was then filtered using trimmatic (v 0.38) software with key parameters:
ILLUMINACIP: TRIM_HOME/adapters/TruSeq3-PE.fa:2:30:10, LEADING:5, TRAILING:5 SLIDINGWINDOWN: 4:20MINLEN:150, to obtain high quality "clean reads".
(b) Using the peanut cultivar Tifruner genome (https:// www.peanutbase.org/download) as the reference genome, an index file was constructed with bowtie2 (v 2.3.5) software, and then "clean reads" of A.dardonoi and A.pusilla were aligned to the Tifruner reference genome, and "clean reads" with neither end aligned to the Tifruner reference genome were filtered out.
(c) 100 ten thousand sequences were randomly sampled from the filtered "clean reads" using the shuf command of Linux, and the "clean reads" obtained by random sampling was subjected to tandem repeat sequence mining using TAREAN software (http:// www.repeatexplorer.org), and a set of "high confidence" tandem repeat sequences was mined.
(d) The tandem repeat sequence was mapped onto the peanut cultivar tifrenner reference genome using B2DSC (http:// mcgb.uestc.edu.cn/B2 DSC), excluding the tandem repeat sequence highly enriched in cultivars; NCBI-BLAST (https:// www.ncbi.nlm.nih.gov /) was used to exclude possible gene sequences for candidate tandem repeats, and the remaining tandem repeats were used for probe design.
(e) A segment with the most conserved A.dardonoi and A.pusilla tandem repeat sequences is selected to intercept a sequence of 30-45bp, and 5'-TAMRA or 5' -FAM is artificially synthesized and modified to obtain H genome specific probes Dard-127 and Pus-187. Probe Dard-127:
FAM-5'-TAAACTATGGTATTTTCATGAGTTTTGAGGCATGCCGGA-3'; (SEQ ID NO: 1) probes Pus-187:
TAMRA-5′-TCACTAGGCATATAATGCCACTCGATGGCGTTGAAACGCGGAGCT-3′(SEQ ID NO：2)。
example 2 design method of peanut wild species B genome specific Probe
(a) The B genome species a.ipaensis was first submitted to illuminea double ended genome sequencing to a depth of 20×. The genome sequencing original sequence was then filtered using trimmatic (v 0.38) software with key parameters: ILLUMINACIP: TRIM_HOME/adapters/TruSeq3-PE.fa:2:30:10, LEADING:5, TRAILING:5 SLIDINGWINDOWN: 4:20MINLEN:150, to obtain high quality "clean reads".
(b) Using the peanut cultivar Tifruner genome (https:// www.peanutbase.org/download) as the reference genome, an index file was constructed with bowtie2 (v2.3.5) software, and then "clean reads" of A.ipaensis were aligned to the Tifruner reference genome, and "clean reads" with neither end aligned to the Tifruner reference genome were filtered out.
(c) 100 ten thousand sequences were randomly sampled from the filtered "clean reads" using the shuf command of Linux, and the "clean reads" obtained by random sampling was subjected to tandem repeat sequence mining using TAREAN software (http:// www.repeatexplorer.org), and a set of "high confidence" tandem repeat sequences was mined.
(d) The tandem repeat sequence was mapped onto the peanut cultivar tifrenner reference genome using B2DSC (http:// mcgb.uestc.edu.cn/B2 DSC), excluding the tandem repeat sequence highly enriched in cultivars; NCBI-BLAST (https:// www.ncbi.nlm.nih.gov) was used to exclude possible gene sequences for candidate tandem repeats, with the remaining tandem repeats being used for probe design.
(e) The most conserved segment of the A.ipaensis tandem repeat sequence is selected to intercept the sequence of 30-45bp, and 5' -TAMRA is artificially synthesized and modified to obtain the B genome specific probe Ipa-163.
Probes Ipa-163: TAMRA-5'-TAGGGTTTATGATTTAGGCTTTAGGGTTTGT-3' (SEQ ID NO: 3). Example 3 use of peanut wild species H genome specific Probe
First, wild species of flowers A.duraensis (A genome), A.digoi (A genome), A.ipaensis (B genome), A.paralagarsis (E genome), A.stenophylla (E genome), A.pusilla (H genome), A.dardonoi (H genome) and A.bat zocoi (K genome) were obtained by a chromosome tabletting method, and they were metaphase-plated by mitosis, frozen at-80℃to remove flakes, dehydrated with absolute ethanol, and air-dried.
Next, 40ml of 2 XSSC buffer (prepared from 0.3M trisodium citrate C) was added to the dye vat 6 H 5 Na 3 O 7 ·2H 2 O and 3M NaCl), 0.1OD of Dard-127 and 0.1OD of Pus-187 probes, and 5. Mu.l of DAPI mother liquor (100. Mu.g/mL DAPI staining solution), the wild-type chromosome slides were placed in a dye vat for staining for 5h, and a fluorescent microscope was photographed to obtain a stained picture (FIG. 1).
As a result, it was found that both probes had no staining signal on the chromosomes of the A genome species A.duraensis and A.digoi, the B genome species A.ipaensis, the E genome species A.paralagariensis and A.stenopylla, the K genome species A.bat. Probes Pus-187 produced red signals (bright spots in the figure) on both the A.pusilla and A.dardonoi chromosomes of the H genome species, indicating that the probes were H genome-specific probes that specifically identified the H genome species. Probe Dard-127 only produced a green signal on the a.dardonoi chromosome (bright spots in the figure), indicating that this probe is a specific probe for the H genome species a.dardonoi, which can specifically recognize a.dardonoi.
Example 4 use of peanut wild species B genome-specific Probe
First, wild species of flowers A.duraensis (A genome), A.digoi (A genome), A.ipaensis (B genome), A.paralagarsis (E genome), A.stenophylla (E genome), A.pusilla (H genome), A.dardonoi (H genome) and A.bat zocoi (K genome) were obtained by a chromosome tabletting method, and they were metaphase-plated by mitosis, frozen at-80℃to remove flakes, dehydrated with absolute ethanol, and air-dried.
Next, 40ml of 2 XSSC buffer (prepared from 0.3M trisodium citrate C) was added to the dye vat 6 H 5 Na 3 O 7 ·2H 2 O and 3M NaCl), 0.1OD of Ipa-163, and 5. Mu.l of DAPI mother liquor (100. Mu.g/mL of DAPI staining solution), the wild-type chromosome slides were placed in a dye vat for staining for 5h, and a fluorescent microscope was photographed to obtain a stained picture (FIG. 2).
As a result, it was found that both probes had no staining signal on the chromosomes of the A genome species A.duranensis and A.diogoi, the E genome species A.paramediciensis and A.stenopylla, the H genome species A.pusilla and A.dardonoi. Probes Ipa-163 only produced red signals (bright spots in the figure) on the B genome species a.ipaensis, K genome species a.bat zocoi chromosomes, suggesting that the probes are B and K genome-specific probes, suggesting that B and K genomes may be more closely related than other genomes.
Sequence listing
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Claims (8)
1. A genome specific probe of a wild peanut species, which is characterized by comprising an H genome specific probe and a B genome specific probe; the H genome specific probes are Dard-127 and Pus-187, the B genome specific probes are Ipa-163, and the specific sequences are as follows:
probe Dard-127:
FAM-5′-TAAACTATGGTATTTTCATGAGTTTTGAGGCATGCCGGA-3′；
probes Pus-187:
TAMRA-5′-TCACTAGGCATATAATGCCACTCGATGGCGTTGAAACGCGGAGCT-3′；
probes Ipa-163: TAMRA-5'-TAGGGTTTATGATTTAGGCTTTAGGGTTTGT-3'.
2. A method of designing a genome-specific probe for a wild species of peanut as claimed in claim 1, comprising the steps of:
(a) Firstly carrying out illuminea double-end genome sequencing on peanut species, wherein the sequencing depth is 20×; then, the genome sequencing original sequence is filtered by utilizing Trimmomatic software, and key parameters are as follows: ILLUMINACIP: TRIM_HOME/adapters/TruSeq3-PE.fa:2:30:10, LEADING:5, TRAILING:5SLIDINGWINDOW:4:20MINLEN:150, to obtain high quality "clean reads";
(b) Using a peanut cultivar Tifrunner genome as a reference genome, constructing an index file by using bowtie2 software, then comparing 'clean reads' to the Tifrunner reference genome, and filtering 'clean reads' of which the two ends are not compared to the Tifrunner reference genome;
(c) Randomly sampling 100 ten thousand sequences from filtered clean reads by utilizing a shuf command of Linux, and utilizing TAREAN software to perform tandem repeat sequence discovery on the clean reads obtained based on random sampling to discover a tandem repeat sequence set of high confidence;
(d) Positioning the tandem repeat sequence onto a peanut cultivar tifrenner reference genome using B2DSC, excluding the tandem repeat sequence highly enriched in cultivars; using NCBI-BLAST to exclude possible gene sequences of candidate tandem repeat sequences, and using the remaining tandem repeat sequences for probe design;
(e) The most conserved segment of the tandem repeat sequence is selected to intercept the sequence of 30-45bp, and 5' -TAMRA or FAM is artificially synthesized and modified to obtain the genome specific probe.
3. The method of designing genome-specific probes for wild peanut species according to claim 2, wherein the peanut species are H genome species a. Dardonoi and a. Pusilla, and the corresponding genome-specific probes are H genome-specific probes Dard-127 and Pus-187.
4. The method of designing a genome-specific probe for a wild peanut species according to claim 2, wherein the peanut species is B genome species a.
5. A method of using the peanut wild type genome specific probe according to claim 1, comprising the steps of:
(a) Chromosome tabletting to obtain metaphase chromosome tablets of root tip cells of diploid wild species with peanut genome A, peanut genome B, peanut genome E, peanut genome H and peanut genome K, respectively, freezing at-80 ℃, removing tablets, dehydrating with absolute ethyl alcohol, and air-drying;
(b) Adding 40mL of 2 XSSC buffer solution, H genome specific probe dry powder or/and B genome specific probe dry powder and 5 mu L of DAPI mother liquor into a dye vat, placing a wild chromosome slice into the dye vat for dyeing 5H, and photographing by a fluorescence microscope; the H genome specific probe dry powder is 0.1OD Dard-127 and 0.1OD Pus-187, and the B genome specific probe dry powder is 0.1OD Ipa-163.
6. The method of using a peanut wild type genome specific probe according to claim 5, wherein the 2 XSSC buffer consists of 0.3mol/L trisodium citrate C 6 H 5 Na 3 O 7 •2H 2 O and 3mol/L NaCl.
7. The method of using a peanut wild type genome specific probe according to claim 5, wherein the DAPI stock solution is DAPI staining solution with a concentration of 100 μg/mL.
8. The method of using a genome-specific probe for peanut wild species as claimed in claim 5, which is specificCharacterized in that the diploid wild species of the A genome, the B genome, the E genome, the H genome and the K genome are respectively A genome speciesA. duranensisGenome of species AA. diogoiSpecies of B genomeA. ipaensisSpecies of E genomeA. paraguariensisSpecies of E genomeA. stenophyllaH genome speciesA. pusillaH genome speciesA. dardonoiSpecies of K genomeA. batizocoi。
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| CN106987590A (en) * | 2017-05-25 | 2017-07-28 | 河南省农业科学院 | One cultivates peanut oligonucleotide probe and its design method and application method |
| CN107130033A (en) * | 2017-05-25 | 2017-09-05 | 河南省农业科学院 | A kind of Peanut genome chromosome sequence figure method corresponding with actual karyotype chromosome sequence number |
| CN109694902A (en) * | 2019-01-28 | 2019-04-30 | 河南省农业科学院 | One cultivates peanut chromosome probe staining kit and its application method |
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| CN104004849A (en) * | 2014-06-09 | 2014-08-27 | 南京农业大学 | Method for quickly establishing metaphase chromosome karyotype of cucumber through genomic in-situ hybridization |
| CN105039542A (en) * | 2015-07-17 | 2015-11-11 | 南京农业大学 | Novel method for painting chromosomes by adopting oligonucleotide probe dye liquor |
| CN106987590A (en) * | 2017-05-25 | 2017-07-28 | 河南省农业科学院 | One cultivates peanut oligonucleotide probe and its design method and application method |
| CN107130033A (en) * | 2017-05-25 | 2017-09-05 | 河南省农业科学院 | A kind of Peanut genome chromosome sequence figure method corresponding with actual karyotype chromosome sequence number |
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